Compositions and methods of using chondroitinase ABCI mutants

ABSTRACT

One aspect of the present invention relates to mutants of chondroitinase ABCI. Such chondroitinase ABCI mutants exhibit altered chondroitin lyase activity or increased resistance to inactivation from stressors including exposure to UV light or heat. Methods of using chondroitinase ABCI mutant enzymes are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/543,749, filed Jul. 6, 2012, now U.S. Pat. No. 8,852,583, issuedOct. 7, 2014, which is a continuation application of U.S. applicationSer. No. 12/757,006, filed Jun. 4, 2010, now U.S. Pat. No. 8,236,302,issued Aug. 7, 2012, which is a continuation application of U.S.application Ser. No. 12/338,207, filed Dec. 18, 2008, now U.S. Pat. No.7,731,956, issued Jun. 8, 2010, which is a divisional application ofU.S. application Ser. No. 11/527,318, filed Sep. 26, 2006, now U.S. Pat.No. 7,485,295, issued Feb. 3, 2009, which claims priority from U.S.Provisional Application No. 60/720,628, filed Sep. 26, 2005; allaforementioned applications being herein incorporated by reference intheir entirety.

BACKGROUND

Proteoglycans, major constituents of the extracellular matrix, are knownto be present in large amounts in glial scar tissue and to inhibitrecovery following spinal cord injuries (Fawcett & Asher, 1999). Enzymesthat are capable of digesting glial scar tissue are an important targetfor the development of spinal cord injury (SCI) therapeutics.Chondroitinase ABCI (EC 4.2.2.4; cABCI) is a bacterial enzyme thatcatalyzes the digestion of sulfated chondroitin and dermatan side chainsof proteoglycans. This enzyme has been shown to promote functionalrecovery after spinal cord injury (Bradbury et al., 2002; Caggiano etal., 2005).

The spinal cord is made up of nerve fibers. Damage to the centralnervous system, including the spinal cord, results in a loss offunction. Depending upon the type of injury to the central nervoussystem, the loss of function may manifest itself in loss of sensory,motor or autonomic function or a combination thereof. Sensory functionsinclude the ability to feel sensations, like pain. Motor functionsinclude the ability to voluntarily move your body. Autonomic functionsinclude involuntary body functions, for example the ability to sweat andbreathe.

The most common types of spinal cord injuries (SCI) include contusions(bruising of the spinal cord) and compression injuries (caused byprolonged pressure on the spinal cord). In contusion and compressioninjuries, a cavity or hole often forms in the center of the spinal cord.Unlike nerve cells, or neurons of the peripheral nervous system (PNS),neurons of the central nervous system (CNS) do not regenerate afterinjury.

Spinal cord injury can be characterized by contusion of the neuraltissue with a resultant decrease or loss of the ability of nerve tissueto properly transmit nerve impulses. The usual cause is due to an impactinjury of some nature, but it may also occur during the manipulation ofthe spinal cord in certain surgical procedures. After a spinal cordinjury in the adult mammal, the inability of axons to regenerate maylead to loss of sensation, loss of motor function and/or loss ofautonomic function, as well as permanent paralysis. One reason thatneurons fail to regenerate is their inability to traverse the glial scarthat develops following a spinal cord injury. The injury-induced lesionwill develop glial scarring, which contains extracellular matrixmolecules including chondroitin sulfate proteoglycans (CSPGs). CSPGsinhibit nerve tissue growth in vitro and nerve tissue regeneration atCSPGs rich regions in vivo.

A number of molecules, and specified regions thereof, have beenimplicated in the ability to support the sprouting of neurites from aneuronal cell, a process also referred to as neurite outgrowth. The termneurite refers to both axon and dendrite structures. The process ofsprouting neurites is essential in neural development and regeneration,especially after physical injury or disease has damaged neuronal cells.Neurites elongate profusely during development both in the central andperipheral nervous systems of all animal species. This phenomenonpertains to both axons and dendrites.

Various polypeptides, especially cell adhesion molecules (CAMs), havebeen known to promote neural cell growth. While early efforts in thisarea of research concentrated on the adhesion-promoting extracellularmatrix protein fibronectin (FN), other polypeptides have also been foundto promote neural growth. For example, U.S. Pat. No. 5,792,743 disclosesnovel polypeptides and methods for promoting neural growth in the CNS ofa mammal by administering a soluble neural CAM, a fragment thereof, or aFc-fusion product thereof. U.S. Pat. No. 6,313,265 discloses syntheticpolypeptides containing the pharmacologically active regions of CAMsthat can be used in promoting nerve regeneration and repair in bothperipheral nerve injuries as well as lesions in the CNS. While helpful,the use of regenerative proteins alone may not be sufficient to effectrepair of a damaged nervous system.

During approximately the past two decades, knowledge of cell adhesionand migration in extracellular matrices (ECMs) at the molecular levelhas expanded rapidly. The action of enzymes and other polypeptides whichdegrade components of the extracellular matrix and basement membranesmay facilitate the events of neural repair by a variety of mechanisms,including the release of bound cytokines and by increasing thepermeability of the matrix, thereby enhancing the mobility of mediatormolecules, growth factors and chemotactic agents, as well as the cellsinvolved in the healing process. For example, U.S. Pat. No. 5,997,863discloses the use of glycosaminoglycans to manipulate cell proliferationand promote wound healing.

Components of the inhibitory CSPGs have been identified as theglycosaminoglycans, chondroitin sulfate (CS) and dermatan sulfate (DS).Removal of these inhibitory molecules would allow neurites to regenerateand reinnervate an area after physical injury or disease, as well as toallow for the recovery of sensory, motor and autonomic functions.

Previous studies have found that chondroitinases can lyse and degradeCSPGs including, CS and DS. One study found that chondroitinase ABCremoved glycosaminoglycan (GAG) chains in and around lesioned areas ofrat CNS in vivo. The degradation of GAGs promoted expression of agrowth-associated protein, GAP-43, indicating an increase in the abilityof treated cells to regenerate. However, this growth-associated proteinis associated with regeneration in peripheral, but not central, nerveinjuries.

Chondroitin sulfates (CS) are sulfated polysaccharides in linear chainsof a repeated dissacharides. They range in molecular weight from about10,000 to over 100,000 Da. Chondroitin sulfate substrates exist indifferent isomers designated by the appended letters A, B, and C(Hoffman et al., 1958). The repeating units are composed of uronic acid(GlcA or IdoA) and galactosamine, and are called galactosaminoglycans,and are one example of the glycosaminoglycans, typically abbreviated asGAG. Although these GAG chain species have different repeatingdisaccharide regions, they are covalently bound through the so-calledlinkage region tetrasaccharide sequence (see below) to the serineresidue in the GAG attachment consensus sequence (Glu/Asp-X-Ser-Gly) ofrespective core proteins. Chondroitin A and C sulfates (ChS-A, ChS-C)are the most abundant GAGs and are found in cartilage, bone and heartvalves. Chondroitin B (ChS-B, or, alternatively, dermatan sulfate) isexpressed mostly in skin, blood vessels, and heart valves.

When chondroitinase bacterial preparations were characterized againstdifferent chondroitin sulfate (ChS) substrates, a series of distinctchondroitinases were discovered: Chondroitinase AC that degrades mostlychondroitin A (ChA) and chondroitin C (ChC) (Yamagata et al., 1968),Chondroitinase B that degrades chondroitin B (ChB) (Michelacci andDeitrich, 1976), Chondroitinase C that acts mostly on ChC (Michelacci YM & Dietrich C P, 1976) and Chondroitinase ABC exhibits specificityagainst all three substrates—ChS-A, ChS-B and ChS-C(Yamagata et al.,1968, Michelacci et al., 1987).

SUMMARY OF THE INVENTION

One aspect of the present invention provides mutants of chondroitinaseABCI.

In preferred embodiments, such chondroitinase ABCI mutants exhibitenhanced activity. In other preferred embodiments, such chondroitinaseABCI mutants exhibit enhanced resistance to inactivation, includinginvactivation from UV or heat exposure. More preferably, thechondroitinase ABCI mutant enzymes are selected from BC6 (SEQ ID NO:1),BE7 (SEQ ID NO:2), BF4 (SEQ ID NO:3). In another preferred embodiment,the chondroitinase ABCI mutant enzymes are selected from BC9 (SEQ IDNO:4), BC7 (SEQ ID NO:5), RD4 (SEQ ID NO:6) and BE11 (SEQ ID NO: 7).

Another embodiment of the present invention is a method of designingmutants of chondroitinase ABCI having altered activity.

Other embodiments of the present invention relate to methods forpromoting neurological functional recovery, including sensory, motor andautonomic function, after central nervous system (“CNS”) injury ordisease.

Further embodiments relate to methods of promoting neuronal outgrowthand the use in treating spinal cord injuries and related disorders ofthe CNS by administering such chondroitinase ABCI mutants.

DESCRIPTION OF THE DRAWINGS

In part, other aspects, features, benefits and advantages of theembodiments of the present invention will be apparent with regard to thefollowing description, appended claims and accompanying drawings where:

FIG. 1 is a bar graph of the chondroitin lyase activity of wild-type,not-inactivated chondroitinase ABCI (normal), wild-type, inactivated (NoEnz) and chondroitinase ABCI mutant enzymes of the present inventionfollowing UV exposure.

FIG. 2 is a bar graph of the chondroitin lyase activity of wild-type,not-inactivated chondroitinase ABCI (normal), wild-type, inactivated (NoEnz) and chondroitinase ABCI mutant enzymes of the present invention.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. Thus, as used herein, theterm “administering”, can include, but is not limited to, providing anenzyme into the CNS or onto the target tissue; providing an enzymesystemically to a patient by, e.g., intravenous injection whereby thetherapeutic reaches the target tissue; providing an enzyme in the formof the encoding sequence thereof to the target tissue (e.g., byso-called gene-therapy techniques). “Administering” a composition may beaccomplished by injection, topical administration, or by either methodin combination with other known techniques.

The term “animal” as used herein includes, but is not limited to, humansand non-human vertebrates such as wild, domestic and farm animals.

The term “improves” is used to convey that the present invention changeseither the appearance, form, characteristics and/or the physicalattributes of the target to which it is being provided, applied oradministered. The change may be demonstrated by any of the followingalone or in combination, including degradation of the CSPGs of thelesioned area of the spinal cord or within the CNS or restoring, inwhole or in part, motor, sensory or autonomic function of the mammal.

The term “inhibiting” includes the administration of a compound of thepresent invention to prevent the onset of the symptoms, alleviating thesymptoms, or eliminating the disease, condition or disorder.

By “pharmaceutically acceptable”, it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding a polypeptide is inserted into a suitableexpression vector which is in turn used to transform a host cell toproduce the protein. Moreover, the phrase “derived from”, with respectto a recombinant gene, is meant to include within the meaning of“recombinant protein” those proteins having an amino acid sequence of anative protein, or an amino acid sequence similar thereto which isgenerated by mutations including substitutions and deletions (includingtruncation) of a naturally occurring form of the protein.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. In part, embodiments of the present invention are directedto the treatment of the central nervous system, such as degradation ofthe CSPGs of the lesioned area of the spinal cord or within the CNS orrestoration, in whole or in part, motor, sensory or autonomic functionof the mammal.

The terms “therapeutically effective amount” or “effective amount”, asused herein, may be used interchangeably and refer to an amount of atherapeutic compound component of the present invention. For example, atherapeutically effective amount of a therapeutic compound is apredetermined amount calculated to achieve the desired effect, i.e., toeffectively treat an injury to the central nervous system. For example,a therapeutic compound comprising a therapeutically effective amount ofchondroitinase which may be purified by a method of the presentinvention and formulated to provide a stable, active enzyme, issufficient to degrade the CSPGs of the lesioned area of the spinal cordor an amount sufficient to restore, in whole or in part, motor, sensoryor autonomic function of the mammal and may result in a regeneration ofneurons in a central nervous system, such as by promoting axonal growthinto an injured area.

The terms “treat,” “treated,” or “treating” as used herein refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) an undesiredphysiological condition, disorder or disease, or to obtain beneficial ordesired clinical results. For the purposes of this invention, beneficialor desired clinical results include, but are not limited to, alleviationof symptoms; diminishment of the extent of the condition, disorder ordisease; stabilization (i.e., not worsening) of the state of thecondition, disorder or disease; delay in onset or slowing of theprogression of the condition, disorder or disease; amelioration of thecondition, disorder or disease state; and remission (whether partial ortotal), whether detectable or undetectable, or enhancement orimprovement of the condition, disorder or disease. Treatment includeseliciting a clinically significant response without excessive levels ofside effects. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment.

The term “vector” refers to a vehicle which can transport the nucleicacid molecules. The nucleic acid molecules encoding the chondroitinasepolypeptide are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector can be a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

Chondroitinase may be obtained from a microorganism that naturallyexpresses a chondroitinase; for example, but not limited to, E. coli,Proteus vulgaris or from the expression of a recombinant protein in ahost cell. The host cell can be a prokaryotic cell (such as E. coli) ora eukaryotic cell (such as yeast, a mammalian cell or an insect cell).

The nucleotide sequence of chondroitinase ABCI is set forth as SEQ IDNO. 8 and the amino acid sequence of chondroitinase ABCI is set forth asSEQ ID NO. 9.

One aspect of the present invention provides mutants of chondroitinaseABCI. In a preferred embodiment, the chondroitinase ABCI mutant enzymesare selected from BC6 (SEQ ID NO:1), BE7 (SEQ ID NO:2), BF4 (SEQ IDNO:3). In another preferred embodiment, the chondroitinase ABCI mutantenzymes are selected from BC9 (SEQ ID NO:4), BC7 (SEQ ID NO:5), RD4 (SEQID NO:6) and BE11 (SEQ ID NO: 7).

Such enzymes may be formulated into pharmaceutical compositions andformulations. Suitable stable formulations and methods of purificationare set forth in co-pending PCT Application No. US2005/017464 filed May18, 2005 entitled “Methods of Purifying Chondroitinase and StableFormulations Thereof” herein incorporated by reference in its entirety.

One aspect of the present invention provides mutants of chondroitinaseABCI. In preferred embodiments, such chondroitinase ABCI mutants exhibitenhanced activity.

In other preferred embodiments, such chondroitinase ABCI mutants exhibitenhanced resistance to inactivation. More preferably, the chondroitinaseABCI mutant enzymes are selected from BC6 (SEQ ID NO:1), BE7 (SEQ IDNO:2), BF4 (SEQ ID NO:3). In another preferred embodiment, thechondroitinase ABCI mutant enzymes are selected from BC9 (SEQ ID NO:4),BC7 (SEQ ID NO:5), RD4 (SEQ ID NO:6) and BE11 (SEQ ID NO: 7).

Another embodiment of the present invention is a method of designingmutants of chondroitinase ABCI having altered activity. The methodcomprises altering the nucleotide sequence or amino acid sequence ofchondroitinase ABCI, expressing the chondroitinase ABCI in a suitablevector and measuring the activity of the mutant enzyme.

In a further embodiment, a stable chondroitinase ABCI enzyme isprovided. The enzyme may exhibit increased resistance to inactivationunder stressed conditions, including exposure to UV light or heat. In apreferred embodiment, the enzyme exhibits increased stability comparedto wild-type chondroitinase ABCI enzyme following a challenge by astress.

A further embodiment of the present invention is a method of treatingcentral nervous system injuries comprising administering achondroitinase ABCI mutant enzyme. In preferred embodiments, thechondroitinase ABCI mutant enzyme is administered in a therapeuticallyeffective amount. In a preferred embodiment, the chondroitinase ABCImutant enzyme is selected from the group consisting of BC6 (SEQ IDNO:1), BE7 (SEQ ID NO:2), BF4 (SEQ ID NO:3), BC9 (SEQ ID NO:4), BC7 (SEQID NO:5), RD4 (SEQ ID NO:6) and BE11 (SEQ ID NO: 7), more preferably,the enzyme is selected from the group consisting of BC6 (SEQ ID NO:1),BE7 (SEQ ID NO:2), and BF4 (SEQ ID NO:3). Such central nervous systeminjuries may include, but are not limited to, spinal cord injuries.

Another embodiment of the present invention is a method promotingneuronal outgrowth comprising administering a chondroitinase ABCI mutantenzyme. In preferred embodiments, the chondroitinase ABCI mutant enzymeis administered in a therapeutically effective amount. In a preferredembodiment, the chondroitinase ABCI mutant enzyme is selected from thegroup consisting of BC6 (SEQ ID NO:1), BE7 (SEQ ID NO:2), BF4 (SEQ IDNO:3), BC9 (SEQ ID NO:4), BC7 (SEQ ID NO:5), RD4 (SEQ ID NO:6) and BE11(SEQ ID NO: 7), more preferably, the enzyme is selected from the groupconsisting of BC6 (SEQ ID NO:1), BE7 (SEQ ID NO:2), and BF4 (SEQ IDNO:3).

Other embodiments of the present invention relate to methods forpromoting neurological functional recovery after central nervous system(“CNS”) injury or disease. In preferred embodiments, the chondroitinaseABCI mutant enzyme is administered in a therapeutically effectiveamount. In particular, the present invention is directed to a method ofutilizing chondroitinase to promote sensory, motor or autonomicneurological functional recovery following injury in or to the spinalcord. Compositions useful in this method include acceptable formulationsof chondroitinase, more particularly sustained release formulations ofchondroitinase. The present invention is also directed to a method ofpromoting neurological functional recovery after a contusion injury tothe spinal cord. The most common types of spinal cord injuries (SCI)include contusions (bruising of the spinal cord) and compressioninjuries (caused by pressure on the spinal cord). In contusion injuries,the most common type of injury, a cavity or hole often forms in thecenter of the spinal cord.

The treatments of the present disclosure deliver an effective amount ofthe mutant or other optional therapeutic agent to the CNS or the injuredsite of the CNS. Such methods may include optionally administering otherchondroitin sulfate proteoglycans, including, but not limited tochondroitinase ABC_(TypeI), chondroitinase ABC_(TypeII), chondroitinaseAC and chondroitinase B or mammalian enzymes with chondroitinase-likeactivity such as Hyal1, Hyal2, Hyal3, and Hyal4, preferably to the CNS,and more preferably to the lesions of the injured area of the CNS.

As is known in the art, chondroitinase polypeptides can be produced bystandard biological techniques or by chemical synthesis. For example, ahost cell transfected with a nucleic acid vector directing expression ofa nucleotide sequence encoding the subject polypeptides can be culturedunder appropriate conditions to allow expression of the peptide tooccur. The chondroitinase polypeptide may be secreted and isolated andfrom a mixture of cells and medium containing the recombinantchondroitinase polypeptide. Aspects of the invention described hereinprovide purification methods wherein the chondroitinase is isolated in apure form that is more stable and active then those methods currentlyused.

Alternatively, the peptide may be retained cytoplasmically by removingthe signal peptide sequence from the recombinant chondroitinase gene andthe cells harvested, lysed and the protein isolated by the purificationmethods described herein.

Chondroitinase may be administered topically, locally or systemically.Topical or local administration is preferable for greater control ofapplication. The chondroitinases, singularly or in combination, can bemixed with an appropriate pharmaceutical carrier prior toadministration. Examples of generally used pharmaceutical carriers andadditives are conventional diluents, binders, lubricants, coloringagents, disintegrating agents, buffer agents, isotonizing fatty acids,isotonizing agents, preservants, anesthetics, surfactants and the like,and are known to those skilled in the art. Specifically pharmaceuticalcarriers that may be used are dextran, sucrose, lactose, maltose,xylose, trehalose, mannitol, xylitol, sorbitol, inositol, serum albumin,gelatin, creatinine, polyethlene glycol, non-ionic surfactants (e.g.polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hardenedcastor oil, sucrose fatty acid esters, polyoxyethylene polyoxypropyleneglycol) and similar compounds. Pharmaceutical carriers may also be usedin combination, such as polyethylene glycol and/or sucrose, orpolyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitanmonooleate (20 E. 0.) is particularly preferred.

A treatment regimen according to the invention may be carried out by ameans of administering a mutant chondroitinase ABCI enzyme of thepresent invention. The treatment regiment may further compriseadministering chondroitinase ABCII, chondroitinase AC and chondroitinaseB or mammalian enzymes with chondroitinase-like activity such as Hyal1,Hyal2, Hyal3, Hyal4 and PH2O to the lesions of the injured area of theCNS. The mode of administration, the timing of administration and thedosage are carried out such that the functional recovery from impairmentof the CNS is enhanced by the promotion of neurite outgrowth.

The effective amount of chondroitinase can be administered in a singledosage, two dosages or a plurality of dosages. Although it is to beunderstood that the dosage may be administered at any time, in oneembodiment, the dosage is administered within 12 hours after injury, oras soon as is feasible. In another embodiment, the dosage isadministered to an injured mammal in one, two or a plurality of dosages;such dosages would be dependant on the severity of the injury and theamount of CSPGs present in the glial scarring. Where a plurality ofdosages is administered, they may be delivered on a daily, weekly, orbi-weekly basis. The delivery of the dosages may be by means of catheteror syringe. Alternatively, the treatment can be administered duringsurgery to allow direct application to the glial scar.

For example, in some aspects, the invention is directed to apharmaceutical composition comprising a compound, as defined above, anda pharmaceutically acceptable carrier or diluent, or an effective amountof a pharmaceutical composition comprising a compound as defined above.

The compounds of the present invention can be administered in theconventional manner by any route where they are active. Administrationcan be systemic, topical, or oral. For example, administration can be,but is not limited to, parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, oral, buccal, or ocularroutes, or intravaginally, by inhalation, by depot injections, or byimplants. Thus, modes of administration for the compounds of the presentinvention (either alone or in combination with other pharmaceuticals)can be, but are not limited to, sublingual, injectable (includingshort-acting, depot, implant and pellet forms injected subcutaneously orintramuscularly), or by use of vaginal creams, suppositories, pessaries,vaginal rings, rectal suppositories, intrauterine devices, andtransdermal forms such as patches and creams.

Specific modes of administration will depend on the indication. Theselection of the specific route of administration and the dose regimenis to be adjusted or titrated by the clinician according to methodsknown to the clinician in order to obtain the optimal clinical response.The amount of compound to be administered is that amount which istherapeutically effective. The dosage to be administered will depend onthe characteristics of the subject being treated, e.g., the particularanimal treated, age, weight, health, types of concurrent treatment, ifany, and frequency of treatments, and can be easily determined by one ofskill in the art (e.g., by the clinician).

Pharmaceutical formulations containing the compounds of the presentinvention and a suitable carrier can be solid dosage forms whichinclude, but are not limited to, tablets, capsules, cachets, pellets,pills, powders and granules; topical dosage forms which include, but arenot limited to, solutions, powders, fluid emulsions, fluid suspensions,semi-solids, ointments, pastes, creams, gels and jellies, and foams; andparenteral dosage forms which include, but are not limited to,solutions, suspensions, emulsions, and dry powder; comprising aneffective amount of a polymer or copolymer of the present invention. Itis also known in the art that the active ingredients can be contained insuch formulations with pharmaceutically acceptable diluents, fillers,disintegrants, binders, lubricants, surfactants, hydrophobic vehicles,water soluble vehicles, emulsifiers, buffers, humectants, moisturizers,solubilizers, preservatives and the like. The means and methods foradministration are known in the art and an artisan can refer to variouspharmacologic references for guidance. For example, ModernPharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman& Gilman's The Pharmaceutical Basis of Therapeutics, 6th Edition,MacMillan Publishing Co., New York (1980) can be consulted.

The compounds of the present invention can be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. The compounds can be administered by continuous infusionsubcutaneously over a period of about 15 minutes to about 24 hours.Formulations for injection can be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents.

For oral administration, the compounds can be formulated readily bycombining these compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by apatient to be treated. Pharmaceutical preparations for oral use can beobtained by adding a solid excipient, optionally grinding the resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include, but are not limited to, fillers such as sugars,including, but not limited to, lactose, sucrose, mannitol, and sorbitol;cellulose preparations such as, but not limited to, maize starch, wheatstarch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as, but not limited to, the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include, but arenot limited to, push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with filler such as, e.g., lactose, binders such as, e.g.,starches, and/or lubricants such as, e.g., talc or magnesium stearateand, optionally, stabilizers. In soft capsules, the active compounds canbe dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In addition,stabilizers can be added. All formulations for oral administrationshould be in dosages suitable for such administration.

For buccal administration, the compositions can take the form of, e.g.,tablets or lozenges formulated in a conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds of the present invention can also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds ofthe present invention can also be formulated as a depot preparation.Such long acting formulations can be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection.

Depot injections can be administered at about 1 to about 6 months orlonger intervals. Thus, for example, the compounds can be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

In transdermal administration, the compounds of the present invention,for example, can be applied to a plaster, or can be applied bytransdermal, therapeutic systems that are consequently supplied to theorganism.

Pharmaceutical compositions of the compounds also can comprise suitablesolid or gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as, e.g., polyethylene glycols.

The compounds of the present invention can also be administered incombination with other active ingredients, such as, for example,adjuvants, protease inhibitors, or other compatible drugs or compoundswhere such combination is seen to be desirable or advantageous inachieving the desired effects of the methods described herein.

The following methods are used to illustrate the various embodiments ofthe present invention. The methods are exemplary methods and are notmeant to limit the invention.

Example 1

The present example illustrates exemplary chondroitinase mutant enzymesof the present invention. All nucleotide and amino acids are indicatedas the wild-type and then the mutant version (Wild-type to Mutant).

Mutant ABCI Nucleotide Amino Acid enzyme sequence sequence BC6 (SEQ IDNO. 1) T1206 to C1206 E403 to G403 C1114 to A1114 W372 to C372 BE7 (SEQID NO. 2) G1925 to T1925 S642 to I642 T2226 to G2226 I742 to M742 BF4(SEQ ID NO. 3) T2160 to A2160 N720 to K720e BC9 (SEQ ID NO. 4) G1238 toA1238 S413 to N413 BC7 (SEQ ID NO. 5) A1468 to G1468 K490 to E490 RD4(SEQ ID NO. 6) T1661 to A1661 L554 to H554 BE11 (SEQ ID NO. 7) A1901 toT1901 D634 to V634 C1935 to T1935 (in wobble position of codon- does notresult in AA change)

Example 2

The present example illustrates the chondroitin lyase activity ofexemplary chondroitinase ABCI mutants according to the present inventionfollowing UV exposure. Mutant chondroitinase ABIC genese were generatedand transformed into bacteria. Bacteria were grown and the mutagenizedchondroitinase expressed. The chondroitinase were then exposed to UVlight and their chondroitin lyase activity measured. As depicted in FIG.1, clone BC6 (SEQ ID NO:1), BE7 (SEQ ID NO:2) and BF4 (SEQ ID NO:3)exhibited greater chondroitin lyase activity following exposure to UVlight as compared to control.

Example 3

The present example illustrates the chondroitin lyase activity ofexemplary chondroitinase ABCI mutants according to the presentinvention. The chondroitinase lyase activity of Clone BC9, Clone BC7,Clone RD4 and Clone BE11 under normal (i.e., non-stressed) conditionswas measured and exhibited decreased activity as compared to control anwild-type chondroitinase ABCI, as depicted in FIG. 2.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontained within this specification.

What is claimed is:
 1. A pharmaceutical composition comprising atherapeutically effective amount of mutant chondroitinase ABC I enzymeselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and apharmaceutically acceptable excipient.
 2. The pharmaceutical compositionof claim 1, wherein the pharmaceutical composition comprises atherapeutically effective amount of mutant chondroitinase ABC I enzymeSEQ ID NO: 1, and a pharmaceutically acceptable excipient.
 3. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition comprises a therapeutically effective amount of mutantchondroitinase ABC I enzyme SEQ ID NO: 2, and a pharmaceuticallyacceptable excipient.
 4. The pharmaceutical composition of claim 1,wherein the pharmaceutical composition comprises a therapeuticallyeffective amount of mutant chondroitinase ABC I enzyme SEQ ID NO: 3, anda pharmaceutically acceptable excipient.
 5. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition comprisesa therapeutically effective amount of mutant chondroitinase ABC I enzymeSEQ ID NO: 4, and a pharmaceutically acceptable excipient.
 6. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition comprises a therapeutically effective amount of mutantchondroitinase ABC I enzyme SEQ ID NO: 5, and a pharmaceuticallyacceptable excipient.
 7. The pharmaceutical composition of claim 1,wherein the pharmaceutical composition comprises a therapeuticallyeffective amount of mutant chondroitinase ABC I enzyme SEQ ID NO: 6, anda pharmaceutically acceptable excipient.
 8. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition comprisesa therapeutically effective amount of mutant chondroitinase ABC I enzymeSEQ ID NO: 7, and a pharmaceutically acceptable excipient.